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Fig. 2 Surface expression of CD117, CD200 and CD371 allows the isolation of seven distinct gammadelta thymocyte populations a , b t-SNE map of TCRdelta + thymocytes coloured by a normalised expression intensities of CD24, CD25, CD73, CD117, CD200 and CD371 or by b FlowSOM automated clustering. Based on flow cytometric analysis of 2 x 5000 TCRdelta + cells pooled from two mice in silico. c Heat map of mean expression intensity of markers within the FlowSOM clusters named A to G. d Distribution of cells within populations A to G shown as a percent of the total TCRdelta + thymocytes from 1- to 28-week-old mice. Bars depict the mean +- SEM ( n = 4, 4, 4, 2, 3 mice). e , f Distribution of cells within populations A to G within the Vgamma1.1 + and Vgamma2 + subsets of TCRdelta + thymocytes visualised as e t-SNE map coloured by FlowSOM clusters as in b , f stacked bar plots. Bars depict the mean +- SEM ( n = 2 mice). g Standard bi-axial TCRdelta + gating of population A to G. Numbers denote the mean +- SEM of total TCRdelta + thymocytes ( n = 2 mice). h , i , j Expression of established surface markers of gammadelta T cells within populations A to G visualised as h representative histograms or i , j representative t-SNE maps ( n = 6 mice). h Dashed lines show background signal by FMO. i , j Colours depict i the location of events assigned to populations A to G within the t-SNE plot or j the normalised expression intensity of each marker

Fig. 5 gammadelta T cells emigrate from the thymus at the C, E and G stages a , b TCRdelta + thymocyte distributions within population A to G after treatment with FTY720 for 5 (5d) or 10 days (10d) visualised using a t-SNE maps and b stacked bar plots of cell numbers within TCRdelta + , Vgamma1.1 + and Vgamma2 + subsets. c Representative CD24 expression within A to G populations after treatment with FTY720 for 5 or 10 days. d , e Expression of CD24 within CD73 - and CD73 + TCRdelta + cells from inguinal lymph nodes after FTY720 treatment for 5 and 10 days as d representative flow cytometry plots and e quantitative bar plots showing the percent of TCRdelta + cells within each population. Data from two independent experiments for both 5- and 10-day treatments ( n = 4 mice per group per time point). Numbers within gates and bar plots depict the mean +- SEM. Statistical analyses were performed using the unpaired two-sided Student's t test

Figure 5 Memory B cells expand in and around pre-existing germinal centres on reactivation, but most cells do not acquire GL7 expression. ( a ) One year after oral priming immunizations, the proportion of NP-specific memory B cells in various tissues was monitored kinetically following a single p.o. challenge immunization with NP-CT. The frequency of NP-specific GFP + cells among total CD19 + B220 + CD138 - B cells at given time points was determined in individual mice ( x ; protocol as in Fig. 3a ). ( b , c ) Flow cytometry analysis of GL7 and IgD expression on GFP + memory B cells following the p.o. challenge. ( b , c ) The proportion of GFP + cells that were GL7 + IgD - was determined using the indicated gates. ( c ) The percentage of GFP + memory B cells that were GL7 + IgD - was low in the PP, while it was two- to threefold higher in the MLN. ( d , e ) Antibody-labelled frozen sections of the PP and MLN were prepared 7 days after the challenge and analysed with confocal microscopy for GL7 (red) and B220 (blue) expression on expanding memory GFP + (green) B cells. ( e ) Close-ups of the PP section marked in the mid panel in d with each individual channel shown in black and white and an overlay in colour in which individual GFP + cells are circled to illustrate that most responding GFP + memory B cells express B220, but not the GL7 marker even if located in a germinal centre. ( f ) Flow cytometry analysis on day 7 of IgA expression in responding GFP + memory B cells in the

Figure 2 The inhibition of cell-autonomous HPSE1 did not affect the surface marker profiles of mouse BM-MSCs. Flow cytometric analysis of MSCs markers, Sca-1, CD73, and CD105, hematopoietic cell marker, CD45, and the endothelial cell marker, CD31 on BM-MSCs by the treatment of OGT2115 (OGT) and the DMSO control. Inhibition of HPSE did not affect these surface markers expressions on BM-MSCs.

Product Specific Information

Description: eBioTY/11.8 recognizes the with CD73 a 69-kDa GPI-anchored cell-surface protein with ecto-5'-nucleotidase activity. Expression on myeloid cells (CD11b) is restricted to the bone marrow. In human CD73 can be induced to secrete a soluble form with IL-2 suggesting a role in mediating activation signals. Differences between human and mouse CD73 have been reported. BALB/c mice have more CD4+CD73+ than CD8+CD73+ while the reciprocal is documented in humans.

CD73 is expressed on a subset of lymphocytes and increases during lymphocyte maturation. Recently it has been found that memory CD4 T cells express and are similar to the uncommitted primed precursor helper cells (Thpp) that can differentiate into Th1 or Th2 cells. Furthermore CD73 has been found on regulatory T cells.

Applications Reported: This eBioTY/11.8 (TY/11.8) antibody has been reported for use in flow cytometric analysis.

Applications Tested: This eBioTY/11.8 (TY/11.8) antibody has been tested by flow cytometric analysis of mouse splenocytes. This can be used at less than or equal to 0.06 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.

eFluor® 450 is an alternative to Pacific Blue®. eFluor® 450 emits at 445 nm and is excited with the Violet laser (405 nm). Please make sure that your instrument is capable of detecting this fluorochome.

Excitation: 405 nm; Emission: 445 nm; Laser: Violet Laser.

Filtration: 0.2 µm post-manufacturing filtered.

Target Information

CD73 (Ecto-5-prime-nucleotidase, 5-prime-ribonucleotide phosphohydrolase) catalyzes the conversion at neutral pH of purine 5-prime mononucleotides to nucleosides, the preferred substrate being AMP. CD73 consists of a dimer of 2 identical 70 kDa subunits bound externally to the plasma membrane by a glycosyl phosphatidyl inositol linkage. CD73 is used as a marker of lymphocyte differentiation. Consequently, a deficiency of CD73 occurs in a variety of immunodeficiency diseases. Other forms of 5-prime nucleotidase exist in the cytoplasm and lysosomes and can be distinguished from CD73 by their substrate affinities, requirement for divalent magnesium ion, activation by ATP, and inhibition by inorganic phosphate. The CD73 gene has been localized to 6q14-q21 by immunofluorescence and a study of a panel of human x mouse hybrids that contained fragments of chromosome 6 as translocations. Defects in the CD73 gene can lead to the calcification of joints and arteries, and intestinal tuberculosis. Two transcript variants encoding different isoforms of CD73 have been found.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.